Inhibitors of kynureninase

ABSTRACT

The present invention provides inhibitors of kynureninase having the formula ##STR1## where X is CHOH, S, SO 2 , SO, SONH 2 , PO 2  H or PONH 2 , R a  and R b , independently of one another are H, a halogen, CF 3  or a small alkyl group having one to three carbon atoms; R 1  is H, NH 2 , NR 6  RT, NO 2 , halogen, CF 3  or a small alkyl group having from one to three carbon atoms, wherein: R 6  and R 7 , independently of one another, are H, a formyl group or a small alkyl group having from one to three carbon atoms with the exception that only one of R 6  or R 7  can be a formyl group; R 2  is OH, H, halogen, CF 3  or a small alkyl group having from one to three carbon atoms; and R 3 , R 4  and R 5 , independently of one another, are H, halogen, CF 3 , NO 2 , NH 2 , or small alkyl group having from one to three carbon atoms. In particular, compounds of this formula in which X is CHOH, S or SO 2  are provided. In compounds of this formula in which X is CHOH, those having the (αS,γS) configuration or the (αR,γR) configuration when R A  or R B  is a hydrogen, are more potent inhibitors of kynureninase. Inhibitors of mammalian kynureninase are of particular use in therapy for certain neurological disorders.

This invention was made through a grant from the National Institutes ofHealth. The United States Government has certain rights in thisinvention.

This is a continuation of application Ser. No. 07/840,408, filed on Feb.24, 1992, now abandoned. Which application is a continuation-in-part ofU.S. Ser. No. 07/689,705, filed Apr. 18, 1991, now U.S. Pat. No.5,254,725.

BACKGROUND OF THE INVENTION

Kynureninases are a group of pyridoxal-5'-phosphate dependent enzymeswhich catalyze the hydrolytic cleavage of aryl-substitutedα-amino-γ-keto acids, particularly L-kynurenine or3-hydroxy-L-kynurenine to give L-alanine and anthranilic acid or3-hydroxyanthranilic acid, respectively (see: K. Soda and K. Tanizawa(1979)Advances Enzym. 49:1-40). Kynureninase is involved in themicrobial catabolism of L-tryptophan via the aromatic pathway. In plantsand animals, a kynureninase is required in tryptophan catabolism and forNAD biosynthesis via quinolinic acid. Quinolinic acid is a relativelytoxic metabolite which has been implicated in the etiology ofneurological disorders, including epilepsy and Huntington's chorea (R.Schwarcz et al. (1988) Proc. Natl. Acad. Sci. USA 85:4079; M. F. Beal etal. (1986) Nature 321:168-171; S. Mazzari et al. (1986) Brain Research380:309-316; H. Baran and R. Schwarcz (1990) J. Neurochem. 55:738-744).Inhibitors of kynureninase are thus important targets for treatment ofsuch neurological disorders.

L-kynurenine (which can also be designatedα,2-diamino-γoxobenzenebutanoic acid) is the preferred substrate ofbacterial kynureninase, which is exemplified by that of Pseudomonasfluorescens (O. Hayaishi and R. Y. Stanier (1952) J. Biol. Chem.195:735-740). The kynureninase of tryptophan metabolism in plants andanimals has a somewhat different substrate specificity with3-hydroxy-L-kynurenine (which can be designatedα,2-diamino-3-hydroxy-γ-oxo-benzenebutanoic acid) being the preferredsubstrate (Soda and Tanizawa (1979) supra).

The mechanism of kynureninases has been the subject of considerableinterest due to the unique nature of this pyridoxal-5'-phosphatedependent reaction. Mechanisms based on redox reactions ((J. B.Longenecker and E. E. Snell (1955) J. Biol. Chem. 213:229-235) ortransamination (C. E. Dalgleish et al. (1951) Nature 168:20-22) havebeen proposed. More recently mechanisms involving either a nucleophilicmechanism with an "acyl-enzyme" intermediate (C. Walsh (1979) "EnzymaticReaction Mechanisms" W. H. Freeman and Co., San Francisco, p. 821; M.Akhtar et al. (1984) "The Chemistry of Enzyme Action" New ComprehensiveBiochemistry, Vol. 6 (M. I. Page, ed.) Elsevier, N.Y., p.821) or ageneral base-catalyzed mechanism (K. Tanizawa and K. Soda (1979) J.Biochem. (Tokyo) 86:1199-1209) have been proposed.

In addition to the physiological reaction, kynureninase has been shownto catalyze an aldol-type condensation of benzaldehyde with incipientL-alanine formed from L-kynurenine to giveα-amino-γ-hydroxy-γ-phenylbutanoic acid (G. S. Bild and J. C. Morris(1984) Arch. Biochem. Biophys. 235:41-47). The stereochemistry of theproduct at the γ-position was not determined, although the authorssuggested that only a single isomer was formed.

J. L. Stevens (1985) J. Biol. Chem 260:7945-7950 reports that rat liverkynureninase displays cysteine conjugate β-lyase activity. This enzymeactivity is associated with cleavage of S-cysteine conjugates of certainxenobiotics to give pyruvate, ammonia and a thiol, for example, cleavageof S-2-(benzothiazolyl)-L-cysteine to give 2-mercaptobenzothiazole,pyruvate and ammonia.

Several reports concerning the relative reactivities of kynurenineanalogs with bacterial kynureninase or rat liver kynureninase aresummarized in Soda and Tanizawa (1979) supra. Tanizawa and Soda (1979)supra reported that a number of ring substituted L-kynurenines, namely:3-hydroxy-, 5-hydroxy-, 5-methyl-, 4-fluoro-, and 5-fluoro-L-kynureninewere substrates of kynureninase of P. fluorescens. These authors alsoreported that dihydrokynurenine (called γ-(o-aminophenyl)-L-homoserinetherein) was a substrate for that kynureninase, yieldingo-aminobenzaldehyde and L-alanine. The K_(m) of dihydrokynurenine wasreported to be 67 μM compared to a K_(m) of 35 μM for L-kynurenine and200 μM for 3-hydroxy-L-kynurenine. N'-formyl-L-kynurenine andβ-benzoyl-L-alanine were likewise reported to be substrates (with K_(m)=2.2 mM and 0.16 mM, respectively) for the bacterial kynureninase.Tanizawa and Soda measured relative reactivity as relative amounts ofL-alanine formed.

O. Hayaishi (1955) in "A Symposium on Amino Acid Metabolism" (W. D.McElroy and H. B. Glass, eds.) Johns Hopkins Press, Baltimore pp.914-929 reported that 3-hydroxy- and 5-hydroxy-L-kynurenine,β-benzoyl-L-alanine and β-(o-hydroxybenzoyl)-L-alanine were substratesfor the bacterial enzyme, but that N'-formyl-L-kynurenine was not asubstrate. O. Hayaishi measured relative reactivities by determining theamount of substrate hydrolyzed.

Tanizawa and Soda (1979) supra reported that S-benzoyl-L-cysteine,L-asparagine and D-kynurenine were not substrates of kynureninase, whileO. Hayaishi (1955) supra reported that β-(p-aminobenzoyl)-L-alanine,β-(o-nitrobenzoyl)-L-alanine, β-(m-hydroxybenzoyl)-L-alanine,3-methoxy-L-kynurenine, β-benzoylpropanoic acid,andβ-(o-aminobenzoyl)propanoic acid do not react with bacterialkynureninase. Kynureninase is reported to act only on L-amino acids (M.Moriguchi et al. (1973) Biochemistry 12:2969-2974).

O. Wiss and H. Fuchs (1950) Experientia 6:472 (see: Soda and Tanizawa(1979) supra) reported that 3-hydroxy-L-kynurenine, L-kynurenine,β-benzoyl-L-alanine, γ-phenyl-L-homoserine, γ-methyl-L-homoserine,2-aminolevulinic acid and α-amino-γ-hydroxypentanoic acid reacted withrat liver kynureninase to produce alanine, whileβ-(o-nitrobenzoyl)-L-alanine did not.

G. M. Kishore (1984) J. Biol. Chem. 259:10669-10674 has reported thatcertain β-substituted amino acids are mechanism-based inactivators ofbacterial kynureninase. Several β-substituted amino acids includingβ-chloro-L-alanine, O-acetyl-L-serine, L-serine 0-sulfate,S-(o-nitrophenyl)-L-cysteine and β-cyano-L-alanine inactivatedkynureninase. These β-substituted amino acids react with kynureninase togive pyruvate and ammonia. However, a portion of the turnovers of theenzyme lead to formation of an inactive enzyme complex.L-S-(o-nitrophenyl)-L-cysteine was described as the "most efficientsuicide substrate at low concentrations" with a K_(i) of 0.1 mM.

Bacterial kynureninase is also strongly inhibited by o-aminobenzaldehyde(K_(i) =6.5 μM, non-competitive inhibition). Several other aromaticshaving "a carboxyl group on the benzene ring and an amino group at theortho-position" including o-aminoacetophenone, anthranilic acido-nitrobenzaldehyde and benzaldehyde were described as inhibitors(Tanizawa and Soda (1979) supra). It was suggested that inhibitionrelates to binding of the formyl group to the portion of the enzyme thatserves as a binding site for the γ-carboxyl of kynurenine. Anthranilateand 3-hydroxanthranilate, the products of the kynureninase reaction,were also reported to inhibit the enzyme (Takeuchi et al. (1980) J.Biochem. (Tokyo) 88:987-994).

J. P. Whitten et al. (1989) Tetrahedron Letts. 30:3649-3652 reported thesynthesis of 2,2-difluoro-α-benzoyl alanine(α-amino-β,β-difluoro-γ-oxobenzene butanoic acid) which is said to be a"potential new inhibitor of kynureninase." Fluoroketone-containingpeptides are described as capable of forming stable hydrates orhemiketals which are "thought to inhibit" proteolytic enzymes as analogsof a tetrahedral transition state. The difluoro compound is described asa competitive inhibitor of kynureninase, but no details of thisinhibition are given in the reference.

The present work is based on a reexamination of the mechanism ofkynureninase catalysis, in particular, through an investigation of thestereospecificity of the retroaldol reaction catalyzed by the enzyme.During the course of this work, the reactivity of dihydrokynurenine withkynureninase was found to be significantly different than had previouslybeen reported. The result of these mechanism and reactivity studies wasthe identification of a class of potent kynureninase inhibitors. Thepresent invention provides kynureninase inhibitors which were designedto be "transition-state analogue" inhibitors.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide means andcompositions for inhibition of kynureninase. The inhibitors of thisinvention are amino acid derivatives of the formula: ##STR2## whereinthe stereochemical configuration at the α carbon is indicated (the sameconfiguration as in L-kynurenine) where X is CHOH, S, SO₂, SO, SONH, PO₂H, or PONH₂, wherein R_(a) and R_(b), independently of one another areH, halogen, CF₃ or a small alkyl group having one to three carbon atoms;R₁ is H, halogen, NH₂, NR₆ R₇, NO₂, CF₃, or a small alkyl having fromone to three carbon atoms; with R₆ and R₇, independently of one another,being H, CH₃ or COH wherein only one of R₆ or R₇ can be COH; R₂ is OH,H, halogen, CF₃ or a small alkyl having from one to three carbon atoms;and R₃, R₄ and R₅, independently of one another, are H, OH, halogen,CF₃, NO₂, NH₂, or a small alkyl group having from one to three carbonatoms.

X is preferably CHOH, S or SO₂ with SO₂ being generally more preferredthan S.

It is preferred that the halogen is fluorine, that R₂ is H or OH, thatR₁ is H or NH₂, and that R_(A), R_(B), R₄ and R₅ are H or fluorine. Itis preferred that R₃ is H, NH₂, NO₂ or fluorine, with H or fluorine morepreferred. It is more preferred that R₁ is NH₂ and R_(A), R_(B), R₃, R₄and R₅ are H.

For inhibition of bacterial kynureninase, it is preferred that R₂ is H.For inhibition of plant and animal kynureninase, it is preferred that R₂is OH.

It is a specific object of the present invention to provide kynureninaseinhibitors which are derivatives of α-amino-γ-hydroxy-γ-hydroxybenzenebutanoic acids of the formula: ##STR3## wherein the stereochemicalconfiguration at the α carbon is as indicated (the same configuration asL-kynurenine), wherein R_(a) and R_(b), independently of one another areH, halogen, CF₃ or a small alkyl group having one to three carbon atoms;R₁ is H, halogen, NH₂, NR₆ R₇, NO₂, CF₃ or a small alkyl group havingfrom one to three carbon atoms, with R₆ and R₇, independently of oneanother, being H, CH₃ or COH, wherein only one of R₆ or R₇ can be COH;R₂ is OH, H, halogen, CF₃, or a small alkyl group having from one tothree carbon atoms; and R₃, R₄ and R₅, independently of one another, areH, OH, halogen, CF₃, NO₂, NH₂, or a small alkyl group having from one tothree carbon atoms. It is preferred that the halogen is fluorine, thatR₂ is H or OH that R₁ is NH₂ or H and that R_(A), R_(B), R₃, R₄ and R₅are H or fluorine. It is more preferred that R₁ is NH₂ and that R_(A),R_(B), R₃, R₄ and R₅ are H.

For inhibition of bacterial kynureninase it is preferred that R₂ is H.For inhibition of plant and animal kynureninase it is preferred that R₂is OH.

It is a more specific object of this invention to provide kynureninaseinhibitors which are α-amino-γ-hydroxy-γ-aryl butanoic acids having thestructure: ##STR4## wherein the stereochemical configuration at the αand γ carbons is as indicated (the configuration at the α carbon beingthe same as in L-kynurenine) and wherein R₁₋₇, R_(A) and R_(B) are asdefined above for formulas I and II. For inhibition of bacterialkynureninase it is preferred that R₂ is H. For inhibition of plant andanimal kynureninase it is preferred that R₂ is OH.

It is a second specific object of this invention to provide S-arylderivatives of L-cysteine which are inhibitors of kynureninase havingthe formula: ##STR5## where the stereochemical configuration at theα-carbon is as indicated (the same as in L-kynurenine) where R₁₋₇, R_(A)and R_(B) are as defined for formulas I, II and III.

It is a further specific object of this invention to provideS-aryl-L-cysteine sulfones which are inhibitors of kynureninase havingthe formula: ##STR6## where the stereochemical configuration at theα-carbon is as indicated (the same as in L-kynurenine) and R₁₋₇, R_(A)and R_(B) are as defined for formulas I-IV.

Salts of the compounds of formulas I-V are considered functionalequivalents thereof with respect to inhibition of kynureninase. Inparticular, pharmaceutically acceptable salts of the compounds offormulas I-V are useful for the methods of the present invention and areuseful in any therapeutic treatment of animals based on the inhibitoryaction of the compounds of formulas I-V.

Inhibitors of the present invention include, among others, ringfluorinated dihydrokynurenines: (αS,γS)- or (αS,γR)-α,2-diamino-γ-hydroxy-4-fluorobenzenebutanoicacid, (αS,γS)- or(αS,γR)-α,2-diamino-γ-hydroxy-4-fluorobenzenebutanoic acid; ringhydroxylated dihydrokynurenines: (αS,γS)- or(αS,γR)-α,2-diamino-γ,5-dihydroxybenzenebutanoic acid; ring methylateddihydrokynurenines (αS,γS)- or (αS,γR)-α,2-diamino-γ-hydroxy-5-methylbenzenebutanoic acid, or ring-substituted (αS,γS)- or(αS,γR)-α-amino-γ,2-dihydroxybenzenebutanoic acid.

Inhibitors of kynureninase also include dihydrokynurenines:(αS,γS)-α,2-diamino-γ-hydroxybenzenebutanoic acid and(αS,γR)-α,2-diamino-γ-hydroxybenzenebutanoic acid;3-hydroxydihydrokynurenines:(αS,γS)-α,2-diamino-γ,3-dihydroxybenzenebutanoic acid and(αS,γR)-α,2-diamino-γ,3-dihydroxylbenzenebutanoic acid anddihydrodesaminokynurenines: (αS,γS)-α-amino-γ-hydroxybenzenebutanoicacid and (αS,γR)-α-amino-γ-hydroxybenzenebutanoic acid.Dihydrokynurenine and dihydrodesaminokynurenine ( see Soda and Tanizawa(1979) Supra p. 32, Table VIII) were previously reported to besubstrates for certain kynureninases. Alternate substrates will act ascompetitive inhibitors toward the "natural" enzyme substrate.Dihydrokynurenine (Tanizawa and Soda (1979) supra) was reported to reactreadily with bacterial kynureninase with a reactivity about 65% that ofL-kynurenine. The dihydrokynurenine employed in that reference wasindicated to be a mixture of the (αS,γS) and (αS,γR) dihydrokynureninediastereomers. It was not disclosed therein and the data given thereindo not suggest that one of the diastereomers (αS,γS) is not a substratefor the kynureninase but acts as a competitive inhibitor of the enzymefor reaction of its natural substrates.

It is a further object of this invention to provide a method ofinhibiting kynureninase in vitro and/or in vivo which comprises the stepof contacting the enzyme with an inhibitory amount of one or more of thecompounds of formulas I-V or salts, particularly pharmaceuticallyacceptable salts, thereof. It is well understood in the art that aprecursor prodrug may be converted in vivo to a therapeutically activedrug. Any such prodrug precursors of the compounds of formulas I-V areencompassed by this invention.

Therapeutic applications of the methods of the present invention relateparticularly to inhibition of animal kynureninases, particularly thoseof mammals. Inhibitors in which R₁ is NH₂ and R₂ is OH are preferred forsuch therapeutic applications.

Compounds of the present invention that are preferred for therapeuticapplications of the methods of the present invention are those that haveminimal toxic or irritant effect toward the target of the therapy. Ifthe inhibitor reacts with kynureninase, it is important that the productof that reaction be substantially nontoxic.

Kynureninases from different sources have different substratepreferences. For example, the preferred substrate of mammaliankynureninase is 3-hydroxy-L-kynurenine rather than L-kynurenine. Ingeneral, for a particular kynureninase, a preferred inhibitor of formulaI-V will possess the phenyl ring substitutions of a preferred substrateof that kynureninase.

DETAILED DESCRIPTION OF THE INVENTION

Kynureninases catalyze the hydrolysis of aryl-substituted γ-keto-α-aminoacids. Kynureninase has been identified and isolated from certainbacteria, fungi, yeasts as well as from mammalian sources. Kynureninasesfrom different sources have been reported to have different substratespecificities. L-kynurenine is the preferred "natural" substrate ofbacterial kynureninase. In contrast for mammalian, yeast and fungalkynureninases, 3-hydroxy-L-kynurenine is the preferred "natural"substrate. This preference for 3-hydroxy-L-kynurenine, as assessed byrelative substrate K_(m) 's, is characteristic of animal and plantkynureninases. The relative affinities of kynureninases for substratesother than L-kynurenine and 3-hydroxy-L-kynurenine can also depend onthe source of the enzyme. Animal and plant kynureninases are sometimescalled 3-hydroxykynureninases. The term kynureninase as used hereinincludes both bacterial, plant and animal kynureninases. Bacterialkynureninases are exemplified by the enzyme isolated from Pseudomonasfluorescens. Mammalian kynureninase is exemplified by the enzymeisolated from mammalian liver, in particular rat liver. A bacterialkynureninase will generally display substrate specificity like that ofthe P. fluorescens kynureninase. Mammalian kynureninase will generallydisplay substrate specificity like that of rat liver kynureninase.Kynureninases, from all sources, catalyze the same types of reactionsand so the mechanisms of the reactions they catalyze should be the same.Differences in affinities for substrates is believed to be associatedwith differences in the substrate binding site.

The present invention provides inhibitors of kynureninase. Some of theseinhibitors are substrates of the enzyme, some are not substrates. Manyof the inhibitors of this invention are competitive inhibitors of theenzyme for their natural substrates L-kynurenine and3-hydroxy-L-kynurenine.

Inhibition, as used herein, refers to inhibition of the hydrolysis ofL-kynurenine and 3-hydroxy-L-kynurenine. Competitive inhibition andnoncompetitive inhibition can be assessed by in vitro methods well-knownin the art. Preferred inhibitors of a particular kynureninase are thosehaving a K_(i) less than or equal to the K_(m) of the preferredsubstrate either L-kynurenine or 3-hydroxy-L-kynurenine for thatkynureninase. In general for competitive inhibitors, it is preferredthat the inhibitor have an affinity equal to or greater than that of thepreferred substrate for the enzyme. The level of inhibition that isachieved is dependent on the concentration of inhibitor in the vicinityof the enzyme. In general, the higher the affinity of the enzyme for theinhibitor, the more potent an inhibitor is. For applications of themethods of inhibition of kynureninase, particularly therapeuticapplications, it is generally preferred to employ high affinity (lowK_(i)) inhibitors to minimize the amount of inhibitor that must beadministered.

Kynureninases are known to catalyze other reactions, for example,cysteine conjugate β-lyase activity. Inhibition of kynureninases canalso be, at least qualitatively, assessed employing in vitro assays forsuch alternate kynureninase activities.

The aldol reaction of L-kynurenine and benzaldehyde catalyzed bykynureninase was found to proceed to give predominantly (80%) the (αS,γR) diastereomer of α-amino-γ-hydroxybenzenebutanoic acid.

The stereospecificity of the aldol reaction, as well as the results ofBild and Morris, Arch. Biochem. Biophys. (1984) 235:41-47, supports ageneral base mechanism for kynureninase, as shown in Scheme I. Thestereospecificity for cleavage of the (4R)-isomer is likely a reflectionof favorable orientation for the active site general base to initiatethe retro-aldol cleavage by proton abstraction (Scheme IA). ##STR7##

The basic group involved is probably the carboxylate that Kishore (1984)supra reported is modified by suicide substrate inhibitors. AlthoughKishore proposed that this carboxylate is responsible for α-protonabstraction, stereochemical studies by Palcic et al., J. Biol. Chem.(1985) 260:5248-5251, found that a α-proton of kynurenine is scrambledbetween the α and β-positions of the L-alanine product, and thus theproton abstraction at the α-C is probably due to a polyprotic base, mostlikely a lysine ε-amino group. In the hydrolysis of L-kynurenine, thesecond general base would be required to assist in hydration of theketone, by abstraction of a proton from a water molecule (Scheme IB).The observed stereochemistry of the aldol-reactions suggests that thewater attacks on the reface of the carbonyl group, giving the(S)-gem-diolate anion. Subsequent rapid collapse of this tetrahedralintermediate is likely and would generate the enzyme-bound enamine ofPLP-L-alanine and anthranilic acid (Scheme IB). In the case of the(4S)-isomer, the carbinol group would mimic this gem-diol tetrahedralintermediate, but is not oriented in a position favorable for theretro-aldol reaction to occur. Thus, this compound is a"transition-state analogue," and would be expected to bind tokynureninase very tightly.

As an extension of these mechanistic studies, the reactivities ofdihydrokynurenine diastereomers were examined. (αS,γ)-Dihydrokynurenine(αS,γ)-α,2-diamino-γ-hydroxybenzenebutanoic acid) was found to be a slowsubstrate for the retro-aldol cleavage reaction catalyzed bykynureninase, while the analogous (αS,γS) diastereomer was unreactive.When these compounds were included in reaction mixtures of enzyme andL-kynurenine, the reaction was strongly inhibited. Analysis of thekinetic data in the presence of various concentrations of thedihydrokynurenines demonstrated that they act as competitive inhibitorswith respect to kynurenine, and the data indicate that(αS,γS)-dihydrokynurenine binds more tightly than does L-kynurenine.This increased affinity of (αS,γS)-dihydrokynurenine is characteristicof mechanism-based, or "transition-state analogue" inhibitors.

The design of the kynureninase inhibitors of the present invention wasbased on the results of the inhibition studies on the diastereomers ofdihydrokynurenine in combination with what is known of substratespecificity of kynureninases.

Although not wishing to be bound by any specific theory, it is believedthat the inhibitors of the present invention represent "transition-stateanalogue" inhibitors of kynureninase in view of the newly proposedmechanism of Scheme I. Based on this proposed mechanismα-amino-γ-hydroxybenzenebutanoic acids having electron withdrawinggroups, including but not limited to, CF₃, halogen, NO₂, CN etc.appropriately substituted on the benzene ring to stabilize the proposed"transition state" will act as inhibitors of the kynureninase.

The kynureninase inhibitors of the present invention can be prepared asexemplified for the preparation of the dihydrokynurenine diastereomersby selective reduction of the keto group of an appropriate γ-keto-aminoacid or by other methods well known in the art. Kynurenines, includingvarious ring-substituted kynurenines, can be prepared by ozonolysis oftryptophans. Alternatively, kynurenine analogs with desired ringsubstitution can be prepared enzymatically from appropriate tryptophansas described in Tanizawa and Soda (1979) supra and O. Hayaishi (1953) inBiochemical Preparations (E. E. Snell, ed.) Vol. 3, John Wiley & Sons,Inc., New York, pp. 108-111. The γ-keto amino acid, β-benzoyl-DL-alaninecan be prepared in several ways (for example, C. E. Dalgleish (1952) J.Chem. Soc. 137-141 and F. M. Veronese et al. (1969) Z. Naturforsch.24:294-300) including amination of β-benzoylacrylate (Tanizawa and Soda(1979) supra). β-Benzoyl alanines having various desired ringsubstitution can be prepared using analogous methods starting withappropriately substituted starting materials. Hayaishi (1955) supra andWiss and Fuchs (1950) supra also provide sources of γ-keto amino acidsuseful for preparation of the compounds of the present invention.β-Benzoyl alanines can be selectively reduced by means known to the artto produce the inhibitors of the present invention.

Similarly, β-substituted γ-keto amino acids can serve as precursors tothe β-(or 2-)substituted γ-hydroxy amino acids of the present invention.Whitten et al. (1990) supra, provides a synthesis of2,2-difluoro-2-benzoyl alanine which can be selectively reduced to giveα-amino-γ,γ-difluoro-γ-hydroxybenzenebutanoic acid. Analogous methodscan be employed to prepare β-substituted, phenyl-ring substitutedγ-hydroxybenzenebutanoic acids of the present invention.

As will be appreciated by those in the art, reduction of a chiralnonracemic γ-keto amino acid, preferably an L-amino acid will generallyresult in a mixture of diastereomers. Techniques are available and wellknown in the art for the separation of diastereomers (HPLC, preparativeTLC, etc.).

S-(nitrophenyl)-L-cysteines (IV (NO₂)), were synthesized by nucleophilicaromatic substitution of fluoronitrobenzenes with L-cysteine in DMF inthe presence of triethylamine (Phillips et al. (1989) Enzyme Microb.Techno. 11:80-83). The unsubstituted S-phenyl-L-cysteine (IV (H)) wassynthesized enzymatically following a procedure by Soda et al. (1983)47(12):2861 (Scheme II). This method involves incubating thiophenol withL-serine in the presence of tryptophan synthase at 37.5° C. for 48 hrs.Reduction of S-(nitrophenyl)-L-cysteines was accomplished by stirringwith Zn dust and acetic acid. The oxidation of thioethers to sulfoneswas achieved by using a procedure described by Goodman et al. (1958) J.Org. Chem 23:1251, with slight modifications. This method produced goodresults when the aryl cysteines were treated with a mixture of formicacid (98%) and hydrogen peroxide (30%). However, when 88% formic acidwas used for this reaction a slightly impure product was obtained andthe yields were also lower. The ease of formation of the sulfone dependson the position of nitro group on the ring. When a nitro group ispresent at the ortho position the completion of reaction took 48 hoursor more, whereas, when there is no nitro group on the ring or when thenitro group is at the 4-position, the reaction is complete in 12 hours.Reduction of the nitro sulfones was performed by catalytic hydrogenationusing acetic acid or formic acid as solvent (Scheme III).

Sulfoxide derivatives (I where X=SO) of the present invention can beprepared from known and readily available starting materials by meanswell-known to the art, for example, by oxidation of correspondingthioethers as described in Example 7 in the presence of a limitingamount of hydrogen peroxide.

Sulfoxamide derivatives of this invention can also be prepared fromknown and readily available starting materials by means well-known tothe art.

Phosphinate and phosphinamide derivatives (I where X =PO₂ H or PONH₂)can be prepared from known and readily available starting materials bymeans well-known in the art, for example, by the Arbuzov reaction(Arbuzov. (1964) Pur Appl. Chem. 9:307-335) or routine modificationsthereof.

The results of competitive inhibition of certain thioether and sulfonecompounds are shown in Table 1. Among all the compounds tested, theunsubstituted S-phenyl-L-cysteine was found to be a very weak inhibitor,with K_(i) value of 0.7 mM, however, its oxidized analog,S-phenyl-L-cysteine sulfone, showed a 180 fold decrease in K_(i) valueto 3.9 μM. Similarly, substitution of an ortho-amino group in theS-(2-aminophenyl)-L-cysteine showed a 318 fold decrease in K_(i) to 2.2μM. The compound which combined both of these structural features,S-(2-aminophenyl)-L-cysteine sulfone turned out to be the most potentinhibitor of kynureninase, with K_(i) of about 70 nM. A similar, butless significant improvement in the activity of the compounds wereobserved by sulfone formation in the cases of 2-nitro,4-nitro and4-amino compounds. The results discussed above on the potent inhibitionby dihydrokynurenines indicate that the kynureninase reaction proceedsvia a gem-diol intermediate. The results of Table 1 indicate that theoxygens on the sulfur mimic the gem-diol tetrahedral transition state inthe reaction of L-kynurenine with kynureninase. Therefore, thesecompounds are additional examples of transition state analogs. Thepresence of amine group and its position on the aromatic ring plays animportant role in the activity of the compound. When the amine group ismoved from the 2-position to the 4-position of the ring, the activity ofthe compound drops 50-fold in case of cysteines and 120-fold in case ofthe respective sulfones. This regiospecificity is expected, since the2-aminophenyl-L-cysteines are closer structural analogues of kynurenine.The presence of the nitro group on the ring decreases the activity ofall the compounds by several fold, possibly due to unfavorable stericinteractions.

As has been described herein, one of the pair of diastereomers in casesin which diastereomers can exist, will be a preferred kynureninaseinhibitor. It will be appreciated, however, that inhibition can beobtained by use of a mixture of the diastereomers. In order to obtainmaximal inhibition for the amount of inhibitor employed, it will bepreferable to maximize the amount of the more inhibitory diastereomer inthe mixture.

                  TABLE 1                                                         ______________________________________                                        Competitive Inhibition of Kynureninase.                                        ##STR8##                                                                     M                  K.sub.i (μM)                                            ______________________________________                                                 ##STR9##      700                                                             ##STR10##     3.9                                                             ##STR11##     100                                                             ##STR12##     23                                                              ##STR13##     2.5                                                             ##STR14##     0.07.sup.1                                                      ##STR15##     --                                                              ##STR16##     12                                                              ##STR17##     140                                                    10.                                                                                    ##STR18##     8.5                                                    ______________________________________                                         .sup.1 This value is an upper limit. K.sub.i here is approximately the        same order of magnitude as the concentration of enzyme in the assay, so       that the steadystate approximation may not apply.                        

EXAMPLES Example 1

Investigation of the Mechanism of Kynureninase-catalyzed adol-reactions.

Bacterial kynureninase was prepared from cells of Pseudomonasfluorescens (ATCC 11250, for example) essentially as described byHayaishi and Stanier (1952) J. Biol. Chem. 195:735-740. Cells were grownon a minimal medium containing 0.1% L-tryptophan as the sole carbon andnitrogen source.

From 100 l of medium, grown for 18 h at 30° C., 230 g of wet cell pastewas obtained. The cells were suspended in 1 l of 0.01M potassiumphosphate, pH 7.0, and disrupted by 2 passages through a Manton-Gaulinhomogenizer. After centrifugation of the cell extract for 1 h at 10000g, the enzyme was partially purified by ion-exchange chromatography onDEAE-cellulose and ammonium sulfate precipitation. The preparation usedin the results of Table 1 exhibited a specific activity of 0.2 μmolmin⁻¹ mg⁻¹.

L-kynurenine and benzaldehyde (in excess) were incubated withkynureninase under the conditions described by Bild and Morris (1984)Arch. Biochem. Biophys. 235:41-47, which is incorporated by referenceherein. The product of this reaction was purified by preparative HPLCand identified as α-amino-γ-hydroxybenzenebutanoic acid. This productwas produced in quantitative yield based on L-kynurenine.

The α-amino-γ-hydroxybenzenebutanoic acid produced in the kynureninasereaction exhibited a negative CD (circular dichroism) extremum at 260nm, with vibronic splitting characteristic of a chirally substitutedbenzoyl alcohol chromophore. Based on a comparison of the CD spectra ofthe product with those of (R)- and (S)-mandelic acids, the predominantchiral product was determined to have the same absolute configuration as(S)-mandelic acid and thus to have the (γR)-configuration. (The terms Rand S are employed as is conventional according to theCahn-Ingold-Prelog rules.) NMR analysis (300 MH_(z) ¹ H) of the productdemonstrates that it is an 80:20 mixture of (αS,γR):(αS,γS)diastereomers of α-amino-γ-hydroxybenzene butanoic acid.

Example 2

Reactivity of Dihydrokynurenine with Kynureninase.

L-kynurenine (from commercial sources) was reduced with NaBH₄ in H₂ O togive dihydrokynurenine [α,2-diamino-γ-hydroxybenzenebutanoic acid]. Theprogress of reaction was monitored by the disappearance of the 360 nm UVabsorption band of L-kynurenine. The reduction resulted in a 60:40mixture of diastereomers. The diastereomers were separated bypreparative HPLC on a 20×250 mm C18 column (Rainin, Dynamax) elutingwith 0.1% acetic acid (5 ml/min). The first peak to elute from the HPLCcolumn was identified by ¹ H NMR analysis to be the(αS,γS)-diastereomer. The second peak to elute was identified by ¹ H NMRanalysis to be the (αS,γR)-diastereomer.

The CD spectra of the separated dihydrokynurenine diastereomers wereconsistent with this identification.

The reactivity of the two dihydrokynurenines with kynureninase in 0.1Mpotassium phosphate buffer, pH 8.0, at 25° was examined. Reaction wasfollowed by the appearance of o-aminobenzaldehyde, as determinedspectrophotometrically by the increase in absorbance at 360 nm (SeeTanizawa and Soda (1979) Biochem. (Tokyo) 86:1199-1209, which isincorporated by reference herein).

The (αS,γR)-dihydrokynurenine diastereomer reacted slowly withkynureninase to produce o-aminobenzaldehyde. No significant reaction ofthe (αS,γS)-diastereomer was detected. Tanizawa and Soda (1979) suprahad reported that dihydrokynurenine reacted with kynureninase with aVmax of about 65% that of L-kynurenine. In contrast, the present workindicates that only the (αS,γR)-diastereomer of dihydrokynureninereacts, only at about 5% of the rate of L-kynurenine. Under theconditions employed and with the bacterial kynureninase prepared asdescribed in Example 1, K_(m) of the reaction of L-kynurenine wasdetermined to be 25 μM. This value is similar to the K_(m) of 35 μM forL-kynurenine obtained by Tanizawa and Soda.

Example 3

Inhibition of Kynureninase by Dihydrokynurenine.

Inhibition of kynureninase by dihydrokynurenine was measured byincluding the potential inhibitor in the enzyme assay mixture (seeExample 1 and Tanzawa and Soda (1979) supra) and determining theapparent Km for L-kynurenine (the preferred substrate of bacterialkynurenine) in the absence and presence of the potential inhibitor.K_(i) values were then calculated using the standard equation:

    (K.sub.m).sub.app =K.sub.m (1+[I]/K.sub.i)

where [I] is the molar concentration of inhibitor and K_(m) =25 μM.

Inhibition of kynureninase by the (αS,γR)- and (αS,γS)-diastereomers ofdihydrokynurenine was examined and K_(i) 's were determined. Bothcompounds strongly inhibited the reaction of kynureninase withL-kynurenine. The K_(i) value for the (αS, γS)-diastereomer was lowerthan for the (αS,γR)-diastereomer. Both compounds were found to becompetitive inhibitors of kynureninase.

Inhibition of mammalian kynureninase can be measured using severaldifferent assays for enzyme activity. Rat liver kynureninase is obtainedfrom homogenization of rat liver, followed by precipitation with (NH₄)₂SO₄, as described by Steven, J. L., J. Biol. Chem. (1985) 260:7945-7950,which is incorporated by reference herein. The activity of rat liverkynureninase was assessed by measurement of the cysteine conjugateβ-lyase activity, as described by Steven (supra), withS-(2-benzothiazolyl)cysteine, a nonphysiological chromophoric substrate.Inhibition of kynureninase by the dihydrokynurenine diastereomers wasassessed with respect to reaction with that substrate.

Both the (αS,γR) and (αS,γS) diastereomers of dihydrokynurenine werefound to inhibit the reaction of rat liver kynureninase. The (αS,γS)diastereomer was found to be the stronger competitive inhibitor withK_(i) under the assay conditions of about 690 μM.

Example 4

Synthesis of S-(phenyl)-L-cysteines (IV (H)).

A mixture containing 1.23 ml (12 mM) of thiophenol, 0.525 g (5 mM) ofL-serine, 10 μM of potassium phosphate buffer, pH 7.8, 0.13 mg (20 nM)of pyridoxal-5'-posphate and 5 mg of tryptophan synthase in a totalvolume of 25 ml was stirred at 37.5° C. After 48 hours the reactionmixture was cooled, the thick white precipitate was filtered and washedwith water and ethanol to give 0.31 g of white crystals ofS-(phenyl)-L-cysteine.

Tryptophan synthase was purified from cells of E. coli CB149 withplasmid pSTB7 containing the trpA and trpB genes from Salmonellatyphimurium, as described by Miles et al. (1989) J. Biol. Chem.264:6280.

Example 5

Synthesis of S-(nitrophenyl)-L-cysteines (IV(NO₂).

To a flask containing 5 g of L-cysteine, 4.47 g of fluoronitrobenzeneand 20 ml of DMF was added 7.84 ml of triethylamine. After stirring atroom temperature for 3-4 hours, the contents of the flask solidifed intoa thick yellow cake. This solid was mixed with 15-20 ml of water andfiltered to give crude S-nitrophenyl-L-cysteine. Recrystalization fromhot water gave lemon yellow crystals of the product.

Example 6

Synthesis of S-(aminophenyl)-L-cysteines (IV(NH₂)).

0.4 g of the S-nitrophenyl compound was dissolved in 50 mL of aceticacid, 2.0 g of zinc dust was added, and the mixture stirred at roomtemperature overnight. After completion of the reaction, the solid wasfiltered on celite and the filtrate was concentrated in vacuo to give anoil. This oil was triturated with water and methanol to give an offwhite solid of the reduced compound.

Example 7

Synthesis of S-phenyl and S-nitrophenyl-L-cysteine sulfones (V(H) andV(NO₂)).

0.65 g of S-phenyl or S-nitrophenyl compound was dissolved in 20 ml of98% formic acid and 4 ml of 30% hydrogen peroxide, and the mixturestirred at room temperature for 12-48 hours, depending on the compoundas discussed above. After completion of the reaction, the solvent wascarefully evaporated in vacuo at 25°-30° C. to give a white solid of thedesired product.

Example 8

Synthesis of S-(aminophenyl)-L-cysteine sulfones (V(NH₂)).

0.4 g of nitrophenyl sulfone was dissolved in 50 ml of formic acid,0.045 g of 10% Pd--C was added, and the mixture hydrogenated for 30minutes. The charcoal was removed by filtration on celite and thefiltrate was concentrated in vacuo to give a light tan oil, which upontrituration with methanol gave a light tan solid of the aminophenylsulfone.

Example 9

Competitive Inhibition of Kynureninase by Compounds (IV and V).

Kynureninase activity was measured at 25° C. by following the decreasein absorbance at 360 nm (ε=-4500 M⁻¹ cm⁻¹). A typical assay mixturecontained 0.4 mM L-kynurenine in 0.04M potassium phosphate, pH 7.8,containing 40 μM pyridoxal-5'-phosphate, at 25° C. The reactions ofS-aryl cysteines and S-aryl cysteine sulfones with kynureninase wereperformed using a spectrophotometric coupled assay with lactatedehydrogenase and NADH, by monitoring a decrease in absorbance due topyruvate formation. A typical assay mixture contained 30 μl lactatedehydrogenase solution (2 mg/ml), 0.1 mM NADH, 40 μMpyridoxal-5'-phosphate, 0.04M Tris.HCl buffer, pH 7.8, with varyingconcentrations of the compounds. The competitive inhibition of thesecompounds was measured by variation of L-kynurenine concentrations atseveral fixed values of the inhibitor. K_(m) and V_(max) values werecalculated by fitting of initial rate data to the Michaelis-Mentenequation with ENZFITTER (Elsevier) on a Z-286 personal computer. KIvalues were determined from the equation:

    v=V.sub.max [S]/(K.sub.m (1+[I]/K.sub.i)+[S]

Results for certain compounds of formulas IV-IX are given in Table 1.

We claim:
 1. A kynureninase inhibitor selected from the group consistingof compounds having the formula: ##STR19## wherein X is SO₂, SO, SONH₂;R_(a) and R_(b), independently of one another are H, a halogen, CF₃ ora small alkyl group having one to three carbon atoms; R₁ is H, NH₂, NR₆R₇, NO₂, halogen, CF₃ or a small alkyl group having from one to threecarbon atoms, wherein:R₆ and R₇, independently of one another, are H, aformyl group or a small alkyl group having from one to three carbonatoms with the exception that only one of R₆ or R₇ can be a formylgroup; R₂ is OH, H, halogen, CF₃ or a small alkyl group having from oneto three carbon atoms; and R₃, R₄ and R₅, independently of one another,are H, halogen, CF₃, NO₂, NH₂ or small alkyl group having from one tothree carbon atoms. except that where X is SO₂ and each of R_(A), R_(B),R₂, R₃, R₄ and R₅ is H, R₁ cannot be H or a small alkyl group havingfrom one to three carbon atoms; where X is SO₂ and each of R_(A), R_(B),R₁, R₃, R₄ and R₅ is H, R2 cannot be CF₃ or a small alkyl group havingfrom one to three carbon atoms; where X is SO₂ and each of R_(A), R_(B),R₁, R₂, R₄ and R₅ is H, R₃ cannot be NO₂, CF₃, a halogen, or a smallalkyl group having from one to three carbon atoms; where X is SO andeach of R_(A), R_(B), R₁, R₃, R₄ and R₅ are H, R₃ cannot be H or a smallalkyl group having from one to three carbon atoms, and when X=SO, andR₂, R₄, R₅ are all H, R₃ cannot be a halogen, NO₂ or a small alkyl grouphaving from one to three carbons.
 2. The inhibitor of claim 1wherein:R_(A) and R_(B), independently of one another are H or F; R₁ isHN₂, H or F; R₂ is OH, H, or F; and R₃, R₄ and R₅, independently of oneanother, are H or F.
 3. The inhibitor of claim 2 wherein R₁ is NH₂. 4.The inhibitor of claim 3 wherein R₂ is H.
 5. The inhibitor of claim 2wherein:R_(A), R_(B), R₃, R₄ and R₅ are H; R₁ is NH₂ or H; and R₂ is OHor H.
 6. The inhibitor of claim 5 wherein R₁ is NH₂.
 7. The inhibitor ofclaim 6 wherein R₂ is H.
 8. The inhibitor of claim 6 wherein R₂ is OH.9. The inhibitor of claim 1 in which X is SO₂.
 10. The inhibitor ofclaim 9 in which R₃ is NO₂.
 11. The inhibitor of claim 9 in which R₁ isNH₂ and R₂₋₅, R_(A) and R_(B) are all H.
 12. A kynureninase inhibitorhaving the formula: ##STR20## wherein X is SO or SO₂ ; wherein R₁ is NH₂; R₂ is OH, and R_(A), R_(B), R₃, R₄ and R₅, independently of oneanother, are H or F.